Team:Penn/Protocols

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Protein Purification

RSB250-A Recipe:

  • 20mM Tris
  • 250mM NaCl
  • 30mM Imidazole
  • 10% Glycerol (v/v)
RSB250-B Recipe
  • 20mM Tris
  • 250mM NaCl
  • 500mM Imidazole
  • 10% Glycerol (v/v)

Induction

  1. Grow a small booster culture in LB Broth overnight (~2-10% of final culture volume) w/ appropriate antibiotics
  2. Innoculate full scale culture & monitor OD600 every 15-30 minutes until OD600 reaches 0.8
  3. At OD600=0.8, induce with appropriate concentration of inducer (e.g. 1mM final concentration of IPTG for lac promoter)
  4. Allow to grow overnight @ 25°C

Pelleting & Sonication

  1. Collect cultures by centrifugation for 30 minutes @4°C & 5000g
    • If sample volume is too large, repeat centrifugation (discarding the supernatant after each centrifugation)
  2. Resuspend pellet in RSB250-A (minimum 5% of culture volume)
  3. Pellet must be completely dissolved (no cell clumps) for efficient lysis.
    • For especially difficult to resuspend pellets, sucking up and ejecting the cell suspension through a large gauge (narrow) needle several times can break up the pellet effectively
  4. Sonicate 5 cycles
    • 1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down
    • Lysis must be done on ice or excessive protein degradation will occur
  5. Spin lysate down @4°C & 20000g for 30 minutes
  6. Transfer supernatant into 15 mL Falcon tubes
  7. Add Ni-Agarose beads (1mL of beads per 100mL of culture volume)
    • Wash beads 2-3 times with equal volumes of RSB250-A
    • Spin down cells @ ~3000 rpm in a microcentrifuge
  8. Rotate overnight in a 4°C cold room
  9. Pour lysate and beads into a chromatography column, allow to empty through gravity flow
    • Retain flow through (FT) on ice for later analysis
  10. Wash with 10 mL RSB250-A two times
    • RSB250-A must be cold
    • Collect wash fractions for later gel on ice
  11. Elute with 1% of culture volume of RSB250-B
    • Collect elution fraction on ice
  12. Eluted protein can be stored at 4°C or -80°C

Thawing Mammalian Cells

  1. Prepare plates with 9mL of appropriate supplemented cell culture media
  2. Take the cells out from the -80 freezer
  3. Shake cells in a water bath at 37 degrees C
    • Do this quickly, DMSO is extremely toxic to cells
  4. Spray this with tons of ethanol, dry with kimwipe, and put this in TC hood
  5. Add 1mL of media to the thawed cells, transfer immediately into plate
  6. Take 1 mL of media from the plate and wash the tubes to get any remaining cells
  7. Incubate overnight in cell incubator
  8. Passage cells 1:2

General Cell Culture

  1. Turn TC hood on to start flow and spray with 70% ethanol 30 min before culturing (see sterile technique protocol).
  2. You want to split cells when you see there is 60-70% confluency (under the microscope, 60-70% of the plate is filled with cells)
  3. Clean hands with 70% EtOH and rub them.
  4. Warm media, PBS, trypsin in a water bath at 37 degrees C for 15 minutes. Be sure not to exceed 15 min.
  5. Warm trypsin to room temperature (check on it periodically to not leave it out too long, because trypsin can degrade once warm).
  6. Aliquot the volume of media, PBS, trypsin into small test tubes depending on how much you need. Put the rest in the refrigerators you got them from
  7. Spray entire cell culture hood surface with 70% EtOH and wipe the cell culture hood
  8. Wipe from back of hood, working your way up to the front
  9. Aspirate your old cell culture media from cell culture plate (tip it to one side).Add 5 mL PBS for a 100mm dish. You can scale this with surface area for a smaller well. This is to wash off any extra proteins that are on the plate, because these can deactivate trypsin.
  10. Aspirate off the PBS
  11. Repeat wash
  12. Add 1 mL 0.25% trypsin for 100mm dishes
  13. Immediately deactivate trypsin with media. Add 9mL for a P100 plate.
  14. Wash plate until cells are suspended & no cell clumps are visible
  15. Seed into new plates in appropriate passage
  16. Label the new plate w/ date, name, dilution, cell type, & passage number (abbreviated P#)
  17. Shake plate on surface left-right and then front-back to evenly spread out the cells
  18. Observe seeded cells and verify they are evenly seeded
  19. Put seeded cell plate back in incubator
  20. Throw old plate away in biohazard
  21. Remove all bottles, place everything but the PBS back into the 4C refrigerator
  22. Spray entire cell culture hood surface
  23. Wipe from back of hood, working your way up to the front
  24. Make sure you change media every 2-3 days because the cells use up the nutrients, glucose, etc.

pDawn-ClyA Blood Agar Plating Protocol

  1. Inoculate a 5mL of LB culture and grow overnight in dark incubator (no light)
  2. Take OD600 reading of culture to determine the number of cfu/mL
  3. Dilute and plate approximately 50 cfu onto a 5% Sheep Blood Columbia Agar plate
  4. Incubate at 25°C for 48 hours with blue light
  5. Observe degree of lysis

Production of Chemically Competent Nissle 1911 Cells

  1. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
  2. Shake vigorously at 37 °C to OD600 ~0.25-0.3.
  3. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice.
  4. Centrifuge the cells for 10 min at 5000 g at 4°C.
  5. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.
  6. Centrifuge the cells as above.
  7. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol.
  8. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.
    • Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.

Low Copy Miniprep (Knight et. al. 2009)

Note: All protocol steps should be carried out at room temperature.

  1. Pellet bacterial cells.
  2. Resuspend pelleted bacterial cells in 250 μL Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube.
  3. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
  4. Add 250 μL Buffer P2 and gently invert the tube 4–6 times to mix.
  5. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
  6. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times.
  7. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
  8. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. compact white pellet will form.
  9. Apply 800 μL supernatant from step 5 to the QIAprep spin column by pipetting.
  10. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  11. Decant the flow through into the column for a second spin through.
  12. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  13. Discard the flow through.
  14. Apply the remaining supernatant from step 5 (usually about 200 μL) to the QIAprep spin column by pipetting.
  15. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  16. Decant the flow through into the column for a second spin through.
  17. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  18. Discard the flow through.
  19. Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
  20. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  21. Discard the flow through.
  22. Heat the elution buffer (either water or buffer EB) to 55°C.
  23. Wash QIAprep spin column by adding 500 μL Buffer PE.
  24. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  25. Discard the flow through.
  26. Wash QIAprep spin column a second time by adding 500 μL Buffer PE.
  27. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  28. Discard the flow through.
  29. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  30. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
  31. Add 20 μL of heated elution buffer (buffer EB) to center of column.
  32. Let stand 1 min.
  33. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.
  34. Add 10 μL of heated elution buffer (buffer EB) to center of column.
  35. Let stand 1 min.
  36. Centrifuge at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge for 60 s.

pDawn Blue Light Induction

  1. Grow an overnight culture of BL21 bacteria containing desired pDawn expression vector in the absence of light
  2. Dilute culture in LB Broth to an OD600 of .01
  3. Grow diluted culture under blue light LED illumination at appropriate temperature for 48 hours
  4. Protein expression can sometime be enhanced by decreasing the temperature

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